Dhasakumar Navaratnam - US grants

DESCRIPTION (from applicant's abstract): Hearing loss affects approximately 35 million Americans. It also affects approximately 35 percent of those 65 and older. The primary cause of hearing loss in this group is a result of a loss of hair cells. Our interest is in understanding the molecular events that determine hair cell function, and how these events are controlled. Hair cells are the primary transducers of sound serving to convert the mechanical energy of sound to a coded a neuronal one. This proposal seeks to understand further how hair cells discriminate between frequencies of sound. Specifically, it aims to extend on the recent experimental evidence relating the primary structure of the calcium gated potassium channel, Slo, to electrical resonance; a phenomenon that allows individual hair cells to discriminate between different frequencies of sound. We will look for proteins that bind to and alter the function of this channel thus altering its ability to respond to particular frequencies of sound. A number of strategies will be used to identify these proteins including degenerate RT PCR based on homology to proteins that re known to bind to Slo, and yeast two hybrid assays utilizing the carboxy [unreadable] terminus of Slo as "bait". Once these proteins have been cloned and identified, immunoprecipitation of tagged fusion proteins will be used to confirm that the interactions are in fact real. Antibodies will be raised against these proteins after expressing them in bacteria. These proteins will be co-expressed with the Slo channel in an attempt to determine how they alter its function(s). The electophysiological properties of different isoforms of the Slo channel will be studied with and without these Slo binding proteins. In addition confocal immuno-fluorescence microscopy will be used to determine how these proteins alter the subcellular distribution of Slo in hair cells. Attempts will be made to determine if these proteins are distributed differentially within the basilar papilla. This will employ both RT PCR of individual fragments of the papilla and immunohistochemistry. Constant surveillance will be maintained to determine if the genomic locus of these proteins (revealed by the genome project) maps to any of the syndromic forms of hearing loss (in view of other potassium channel defects causing syndromic hearing loss).

DESCRIPTION (provided by applicant): Hearing loss affects approximately 25 Million Americans. It also affects approximately 35% of those 65 and older. The primary cause of hearing loss in this group is a result of a loss of hair cells. Our interest is in understanding the molecular events that determine hair cell function, and how these events are controlled. We anticipate that such an understanding will allow rational therapies to be developed to treat hair cell loss. Hair cells are the primary transducers of sound serving to convert the mechanical energy of sound to a coded a neuronal one. In this proposal we are seeking to understand the molecular basis of the large conductance potassium channel as it affects hair cell function. Specifically, we are trying to elucidate the molecular determinants of how these channels bring about electrical tuning, a mechanism of frequency discrimination. We are also interested in determining the molecular basis of how they are clustered and co-localized with the voltage gated Ca channel at the basolateral surface of hair cells. The loss of the large conductance potassium channel in mice results in progressive hearing loss. We propose to extend previous work that has identified variations in the primary structure of the protein, and now seek to determine how interacting proteins influence its function. We have already isolated several proteins from the cochlea that interact with a limited portion of this channel using the yeast 2 hybrid technique. We will now determine how these interacting proteins affect the kinetic properties of this channel. We will also determine how these proteins affect it basolateral sorting, clustering and co-localization with voltage gated Ca channels. In addition, we will extend our successful yeast 2 hybrid approach to identify other proteins that interact with entire BK channel. These proteins too will be subject to similar assays. For these experiments we wilt use the chick as a model and will use a modular set of assays that we have developed to test their significance in affecting these physiological processes.

DESCRIPTION (provided by applicant): With the identification of prestin as the elusive lateral membrane motor protein of the outer hair cell (OHC), we are faced with the possibility of understanding how this single molecule can affect the mammal's exquisite sense of hearing. To that end, we have focused our interest on determining what protein structures may give rise to the motor's known biophysical attributes, including its temperature, tension, and voltage dependence. We hypothesize that these molecular activities arise and/or are influenced by interactions of prestin's intracellular C and N termini with other intracellular proteins and anions, and possibly by multimeric interactions, as well. We propose to target a focused set of aims, including 1) determine the contribution of prestin's C and N termini to prestin's signature biophysical attributes, 2) determine prestin's trafficking route in a prestin cell line that we have developed, and 3) determine what structures are different between prestin (slc26a5) and its closet family member slc26a6 that account for prestin's nonlinear capacitance. In order to reach these goals, we will employ a host of electrophysiological, molecular biological and biochemical methods. We believe that the information that we obtain through these studies will aid in understanding how the OHC enables us to hear so well and in turn how we might combat pathologies of the OHC that afflict millions.

DESCRIPTION (provided by applicant): The proposal seeks to bring a molecular understanding of how large conductance calcium activated potassium (BK) channels, which are important for hearing affect hair cell function using mouse and chicken hair cells as a model system. The channels consist of the Slo protein and a variable number of associated proteins The proposal seeks to determine how BK channels are localized at the basolateral surface of hair cells. In particular the proposal seeks to ascertain how Slo trafficking in hair cells occur ad tests the hypothesis that Slo in hair cells uses the basolateral secretory pathway in polarized epithelial cells. The role of phosphorylation and associated proteins in bringing about basolateral sorting will be explored. The project will also determine how splicing, phosphorylation and Slo associated proteins affect BK kinetics attempting to relate these changes to those in native hair cells.

With the identification of prestin, an anion transporter (SLC26) family member, as the elusive lateral membrane motor protein of the outer hair cell (OHC), we are faced with the possibility of understanding how this single molecule can effect the mammal?s exquisite sense of hearing. We propose to target a focused set of aims, including 1) determining the role of a leakage conductance in prestin, distinct from its transporter pathway, in hearing and defining its structural basis; 2) determine the role of anion binding residues, and the influence of mechanical load in governing prestin?s frequency dependence; simultaneous measures of sensor charge movement (NLC), electromotility and evoked-forces are planned and 3) determine the structural components of pillars that link prestin to the underlying cytoskeleton, and confirming the importance of such links in hearing. In order to reach these goals, we will employ a host of genetic, electrophysiological, molecular biological and biochemical methods. We believe that the information that we obtain through these studies will aid in understanding how the OHC enables us to hear so well and in turn how we might combat pathologies of the OHC that afflict millions.